Abstract Mg2+ plays an essential role in a variety of cellular functions, as enzymatic cofactor, regulator of lipid-derived second messengers, promoter of genomic stability and marker for bacterial pathogenesis, among other functions. In this proposal we will investigate the molecular basis for gating and divalent permeability of the Mg2+ channel CorA, the primary Mg2+ uptake system for Eubacteria and Archaea and a close homolog for the mitochondrial Mg2+ importer Mrs2p. The structures of the CorA orthologs from Thermotoga maritima and Methanocaldococcus janaschii have been recently determined, revealing a funnel-shaped homopentamer with 2 transmembrane (TM) helices and a large, mostly helical extracellular region. The overall, long-term goal of this project is to understand the molecular mechanism of Mg2+ translocation and homeostasis in prokaryotic. Although the recent determination of the CorA crystal structures and our own functional and spectrospcopic work during the past funding period have dramatically improved our knowledge of this class of molecules, a number of mechanistic questions remain to be solved. This is particularly true for the molecular events underlying ion selectivity and permeation as well as Mg2+ -driven channel gating. In this respect, we plan to experimentally address several fundamental questions: How does CorA select for Mg2+ against a host of monovalent and divalent cations? How does Mg2+ drive gate closing? What is the physical basis of the energy transduction steps, starting with Mg2+ binding and culminating in protein motion? What are the mechanisms that underlie inward rectification? The approach we plan to pursue combines single particle cryo-EM, reporter-group spectroscopic techniques (spin labeling/ CW and DEER EPR,) X-ray crystallography and electrophysiological methods with classical biochemical, genetic and molecular biological procedures. Functional studies will be targeted to understand the physical basis of ion selectivity and energy transduction in CorA. Information on the topology, secondary, and tertiary structure of CorA and structurally similar orthologs will be obtained from cryo-EM and EPR analysis of spin labeled mutants. This data will be computationally interpreted to generate models of the different stages of the gating pathway, w i t h multiple conformers contributing to a highly dynamic open conformation. This proposal aims to continue a new experimental avenue that will contribute to the understanding of Mg2+ homeostasis in prokaryotes with particular emphasis on the mechanisms of ion translocation and gating, and ion selectivity.